Clinically unscreened vasculo-glial-neuronal coupling is critical for physiological brain function

虽然许多努力致力于了解与活动诱发的变化有关的机制， 脑血流（CBF）即功能性充血和神经血管耦合，对脑血流的作用了解较少。 控制基础CBF和静息神经元活动的过程。考虑到慢性脑灌注不足 导致认知障碍，我们的团队有兴趣研究细胞机制， 稳态血管紧张度的变化以及因此灌注的变化影响静息神经元功能。我们的中央 假设定义生理性血管-神经胶质-神经元偶联（VGNC）的组成性机制是 在疾病中受损。使用多学科的方法，我们将解决以下三个目标：目标1：测试 疾病中异常星形胶质细胞Ca 2+信号传导损害VGNC的假说。组成型星形胶质细胞 将灌注状态整合到大脑中，并通过释放胶质递质信号，调节静息神经元 相应的活动。VGNC的损伤使大脑处于谷氨酸兴奋性毒性，炎症和 氧化应激使用GLAST-CreERT 2; R26-lsl-GCaMP 3小鼠（体内和体外），我们将测量星形胶质细胞的 管腔内↑或↓引起的实质小动脉血管反应性变化引起的钙事件 来自对照和脑低灌注（双侧颈总动脉狭窄）的脑切片中的流量/压力 小鼠将使用药理学方法定义关键信号机制（即P2 Y1和TRPV 4通道） 介导VGNC。目标2。检验血管反应性的变化与 伴随着静息皮质锥体神经元活动的变化。最佳能量平衡要求 神经元活动与血液灌注相匹配。使用体内和体外方法，我们将 确定压力/流量实质小动脉直径变化如何影响对照组静息神经元活动 小鼠和使用血管紧张素II依赖性高血压和脑血管疾病模型 低灌注体外：测量小动脉直径、神经元膜电位、放电率和 在血流动力学激发之前、期间和之后获得突触电流（例如，↑或↓流量/压力） 诱发加压PA。体内：静息神经元活动对血液中系统诱发变化的反应 将对压力进行评估。目标3。检验假设，即血管反应性的变化，通过 星形胶质细胞Ca 2+依赖性通路，GABA能中间神经元调节皮质神经元网络。 同时使用脑实质小动脉直径变化和电生理神经元活动 记录，我们将确定压力/流量诱发的实质小动脉血管反应性变化的影响 对皮质GABA能中间神经元功能和神经元网络有影响。具体来说，我们将确定 GABA能中间神经元亚型在VGNC期间驱动神经元网络反应， 反应需要星形胶质细胞Ca 2+的变化作为中间步骤，以及中间神经元的反应是否 在疾病条件下改变。 1

While much effort has been devoted to the understanding of mechanisms linked to activity-evoked changes in cerebral blood flow (CBF) namely, functional hyperemia and neurovascular coupling, less is understood about the processes controlling basal CBF and resting neuronal activity. Considering that chronic brain hypoperfusion contributes to cognitive impairments our group is interested in studying the cellular mechanisms by which changes in steady-state vascular tone, and thus perfusion, affect resting neuronal function. Our central hypothesis is that constitutive mechanisms defining physiological vasculo-glial-neuronal coupling (VGNC) are impaired in disease. Using a multidisciplinary approach we will address the following three aims: Aim1: Test the hypothesis that aberrant astrocytes Ca2+ signaling in disease impairs VGNC. Astrocytes constitutively integrate perfusion status to the brain and, through the release of gliotransmitter signals, adjust resting neuronal activity accordingly. Impairments in VGNC places the brain at risk for glutamate excitotoxicity, inflammation and oxidative stress. Using GLAST-CreERT2; R26-lsl-GCaMP3 mice (in vivo and in vitro) we will measure astrocytic Ca2+ events in response to parenchymal arteriole vascular reactivity changes evoked by ↑or↓ in lumen flow/pressure in brain slices from control and cerebral hypoperfused (bilateral common carotid artery stenosis) mice. A pharmacological approach will be used to define key signal mechanisms (i.e. P2Y1 and TRPV4 channel) mediating VGNC. Aim2. Test the hypothesis that changes in vascular reactivity are directly associated with changes in resting cortical pyramidal neuron activity. Optimal energy balance requires that the degree of neuronal activity be properly matched with blood perfusion. Using in vivo and in vitro approaches, we will determine how pressure/flow parenchymal arteriole diameter changes impact resting neuronal activity in control mice and in models of vascular disease using Angiotensin II-dependent hypertension and cerebral hypoperfusion. In vitro: measurements of arteriolar diameter, neuronal membrane potential, firing rates and synaptic currents are obtained before, during and after a hemodynamic challenge (e.g. ↑or↓ flow/pressure) evoked to pressurized PA. In vivo: resting neuronal activity in response to systemic-evoked changes in blood pressure will be assessed. Aim3. Test the hypothesis that changes in vascular reactivity recruit, via an astrocyte Ca2+-dependent pathway, GABAergic interneurons to regulate cortical neuronal networks. Using simultaneous parenchymal arteriole diameter changes with electrophysiological neuronal activity recordings we will determine the effect pressure/flow-evoked parenchymal arteriole vascular reactivity changes has on cortical GABAergic interneuron function and neuronal networks. Specifically, we will identify the GABAergic interneuron subtype driving neuronal network responses during VGNC, whether interneuron responses require astrocyte Ca2+ changes as an intermediate step and whether interneuron responses are altered in disease conditions. 1